LC-MS/MS Analysis

Instrumentation

Quantification of PAESe was performed using an Agilent Technologies, Inc., XCT Ultra Plus ion-trap mass spectrometer with an ESI interface (Santa Clara, CA), following chromatographic separation using an Agilent 1100 HPLC system consisting of an autosampler and binary pump. Solvents were vacuum filtered through a 0.45 m nylon membrane (Gelman Sciences, Ann Arbor, MI, USA), and degassed prior to use. Separation was achieved by automated injection of 20 L samples onto a reversed phase C18 guard and analytical column Zorbax Eclipse Plus C18 column (100 × 4.6 mm, 3.5 μm, AgilentTechnologies) equipped with a Phenomenex (Torrance, CA) Security Guard C18 guard column (4.0 × 3.0 mm) under isocratic conditions at a flow rate of 250L/min. An additional in-line pre-filter (MacMod Analytical, Inc., Chadds Ford, PA, USA) was inserted before the guard column. The column effluent was introduced into the mass spectrometer using an electrospray ion-source. High purity nitrogen was used as the curtain and collision gas. Data analysis was performed using ChemStation, v. 6.1 (Agilent Technologies, Santa Clara, CA).

MS Optimization

PAESe and FPAESe, 4-fluoro-phenyl-2-aminoethyl selenide - used as internal standard (I.S.), were dissolved at a concentration of 0.5 g/mL in mobile phase consisting of MeOH and 0.2% (v/v) formic acid (50:50, v/v) and filtered through a 0.45m nylon filter (Gelman Sciences, Ann Arbor, MI). Samples were introduced into the mass spectrometer via the ionization source at a flow rate of 6.0-10.0L/min using a syringe pump (kdScientific Inc., New Hope, PA). Parent compounds were identified from total-ion chromatograms (TIC) acquire in positive ion-mode over the range of 100-1000 m/z. Product-ion chromatograms (XIC) were acquired for each parent (precursor) ion (i.e., PAESe and FPAESe) over the range of 50-200 m/z. A unique ion-pair (precursor and product ion) was identified for each compound. Mass spectrometry parameters for individual ion-pairs were optimized initially using manufactures software and then refined in the manual tune mode.Desolvation gas was set at 350 °C with a flow rate of 9.0 L/min and nebulizer pressure of 40.0 psi. The optimal capillary voltage and capillary exit voltage were 4000 V and 100 V, respectively, with a skimmer voltage of 40 V.

Preparation of Standards and Quality Control Samples

Stock solutions of PAESe and FPAESe were weighted individually and dissolved in mobile phase to prepare stock solutions for use as standards, quality control (QC) or I.S. at 100 μM. To ensure similar sample matrices, calibration curves were prepared by serial dilution of a PAESe stock solution into mobile phase. A 10 L volume of each was transferred into a vial containing 10 L FPAESe (100 M), 50 L blank plasma and 180 L ACN to achieve a plasma concentration range of2,000, 1,600, 1,000, 400, 200, 160, 100, 50 and 20 nM.Quality control (QC) samples were prepared in triplicate at60, 300 and 600 nM, as described above. The FPAESe stock solution was diluted in mobile phase to achieve a final concentration of 400 nM. All standard and QC samples were prepared freshly for each analysis.

Sample Preparation

Plasma samples (50 μL)were deproteinated with ice-coldACN (200 μL),to achieve a final plasma/organic solvent ratio of 20% (v/v), and vortexed to precipitate plasma proteins. A 10 μL volume of freshly prepared FPAESe solution was diluted in mobile phase then spiked into the mixture of ACN and plasmato achieve a final concentration of 400 nM.The mixture was cooled in an ice-water bath for 1 hr, centrifuged at 1,200 × g for 10 min at 4°C. The deproteinized supernatant was dried with a stream of filtered nitrogen and the residues were reconstituted with 100 μL of mobile phase by vortex mixing, resulting in a 50% dilution of samples and standards.Reconstituted samples were transferred into low volume glass vial inserts forLC-MS/MS analysis.

Assay Performance and Validation

Standard and quality control samples were prepared by extraction of pooled plasma from untreated animals, described above. The specificity and selectivity of method was determined based on MRM response of drug-free plasma and plasma spiked with PAESe or FPAESeto monitor specific ion-transitions and the retention time of the analytes of interest. Standard curves for PAESe were constructed by calculating the ratio of the analyze peak area to that of the I.S., and plotting the ratio versus the theoretical concentrations. Data was fit using weighted least squares; the inverse of the variance (1/x2) of the data was used as a weighting factor. The standard curve was considered acceptable if thecalculated accuracies of >90% of the standards were within15% of their theoretical value and no systematic deviationsover the linear range were observed. The LLOQ was determined for the lowest standard withan accuracy within ±20% of the theoretical concentration with a coefficient of variation(CV) < 20% and a signal intensitythat was at least 5 times greater than the baseline noise (S/N > 5). The absolute sensitivity was determined experimentallyby calculating the amount of analyte that wasapplied to the column at the LLOQ, given the sample injection volume (20 L).Intra-day and inter-day variation and accuracies weredetermined by analyzingQC samples, in triplicate, prepared at3different concentrations independently within 24 hr or at 3 different concentrations each day over 3 or more separate days within a 7 day period.

Sample stability was determined under various temperature-dependent storage conditions. Two different concentrations of PAESe (0.5 and 1.0 μM) in triplicate were stored at room temperature, -20 or -80°C for 4 weeks.For freeze-thaw stability, the samples were stored at -20°C, and were then thawed at room temperature for 24 hr followed by refreezing at -20°C for 3 cycles.Stability was determined by comparing the measured concentration of each sample to theoretical concentration of freshly prepared samples; a percent change or CV < 15 % was considered significant.

Validation of LC–MS/MS Method

The representative total ion-chromatogram (TIC) and product ion-chromatogram (XIC) of plasma sample spiked with PAESe or FPAESe are shown in Fig. S2 and S3.A unique precursor/product ion-pairs at 184.0→154.7m/z for PAESe and 204.0→ 174.7m/z for I.S. were identified.A retention time of 3.6 min for both PAESe and FPAESe with atotal chromatographic time of 5.0 min was achieved.Neither cross-talk, nor endogenous interferencearound the retention times of PAESe and FPAESe in the blank plasma were observed.

The LLOQ of PAESe was 50 nM with inter-day accuracy of 80.8% and a precision of 18.2%, which was sufficient for the purpose of this study. A good linearity was shown over the entire concentration range with a correlation coefficient (r2) of 0.995 or better. Intra- and inter-day accuracies and precision of the QCsamples were within an acceptable range of±15% of their theoretical concentrations(Table SI). No significant degradation at 20 (room temperature), -20 or -80 oC was observed; all stability samples ranged from 98 to 110% of their theoretical concentrations with CV of 3.43 to 13.0%.

Table SI. Intra-day and Inter-day accuracy and precision for PAESe

QC (nM) / Intra-day assay / Inter-day assay
Accuracy
(%) / Precision (CV%) / Accuracy
(%) / Precision (CV%)
60 / 101 / 14.0 / 102 / 14.9
300 / 103 / 8.5 / 95.9 / 14.4
600 / 93.3 / 10.4 / 95.9 / 9.8

Legends for Supplementary Figures

Fig. S1. Encapsulation Efficiency according to the drug:lipid ratio Encapsulation efficiency of SSL-PAESe was determined by altering drug:lipid ratio. Encapsulation efficiency was decreased with increasing drug:lipid ratio.

Fig. S2. Total ion chromatogram (a) PAESe; (b) FPAESe(I.S.)Blank rat plasma spiked with PAESe or FPAESe of 300 ng/mL was deproteinated with ACN to remove plasma protein.The mixture was cooled on ice for 1 hr, and then was centrifuged at 1,200 × g for 10 min at 4°C. The supernatant transferred into new tubes was evaporated with a stream of dry nitrogen, followed by reconstitution with mobile phase to inject into the LC-MS/MS system for analysis. The retention time was 3.6 min for both PAESe and FPAESe.

Fig. S3. Product ion chromatogram (a) PAESe; (b) FPAESe (I.S.) Blank rat plasma spiked with PAESe or FPAESe of 300 ng/mL was prepared as described in Fig. S2,MRM mode was applied to monitor specific ion transitions of PAESe and I.S. The maximum signal was detected as the precursor ions at m/z 184.0 and m/z 204.0 for [M+H]+of PAESe and I.S., respectively. The most intense signal was determined at m/z 184.0→154.7 for PAESe and m/z 204.0→ 174.7 for I.S.as precursor/product ion-pairs.

Fig. S1.

Fig. S2.

Fig. S3.